The capacity of certain porous support particles to cause selective retardation based on either size or shape is well known. Such particles are used in chromatographic separation techniques, for example gel filtration, to separate biological macromolecules, e.g. proteins, DNA, RNA polysaccharides and the like. The sieving particles are characterized by the presence of a microporous structure that exerts a selective action on the migrating solute macromolecules, restricting passage of larger particles more than that of the smaller particles. Thus, the utility of sieving lies in the capacity of the particles to distinguish between molecules of different sizes and shapes.
Affinity chromatography is a chromatographic method used for the isolation of proteins and other biological compounds. This technique is performed using an affinity ligand attached to a support particle and the resulting adsorbent packed into a chromatography column. The target protein is captured from solution by selective binding to the immobilized ligand. The bound protein may be washed to remove unwanted contaminants and subsequently eluted in a highly purified form.
Good separation using chromatography techniques depends on the size of particles, the size distribution of particles and the porosity of the particles. The beads, once packed into a column, should be of a high strength in order to support the liquid flow rates observed during purification and column regeneration. The effect of polymer concentration and other preparation parameters on agarose particle porosity and strength are presented in S. Hjertén and K. O. Eriksson, Analytical Biochemistry, 137, 313–317 (1984), herein incorporated by reference. Additional fundamental information is presented in Studies on Structure and Properties of Agarose, A. S. Medin, pH.D. Thesis, Uppsala, 1995, herein incorporated by reference. The description of chemical additives that help to improve the agarose particle porosity are found in M. Letherby and D. A. Young, J. Chem. Soc., Faraday Trans. 1, 77, 1953–1966 (1981) and in M. Tako and S. Nakamura, Carbohydrate Research, 180, 277–284 (1988), both herein incorporated by reference.
Many particle formation methods and apparatus have been developed using centrifugal action to divide a liquid or into droplets or particles. Rotary atomizer machines in general are discussed in the text Spray Drying Handbook, K. Masters, Fifth edition, Longman Scientific & Technical, Longman Group UK Limited, herein incorporated by reference. Other relevant references related to atomization are Atomization and Sprays, A. Lefebvre, Hemisphere Publications, 1989 and Liquid Atomization, L. Bayvel and Z. Orzechowski, Taylor and Francis, 1993, both herein incorporated by reference. A fundamental theory used in the present invention is known as “spray congealing”, based on spray drying principles with the exception that solidification is the objective instead of drying. Traditional emulsion based methods for agarose bead preparation are described in, for example, Studies on Structure and Properties of Agarose, A. S. Medin, pH.D. Thesis, Uppsala, 1995 and in “The Preparation of Agarose Spheres for Chromatography of Molecules and Particles”, Biochimica et Biophysica Acta, 79, 393–398 (1964).
The particle size distribution produced by known apparatus and methods require further sorting steps or procedures in order to select particles of uniform size required for chromatography. The additional sorting steps introduce further costs that could be avoided if the factors determining size distribution of the particles and operating variables are closely controlled. Without additional sorting steps, the products manufactured by conventional rotary atomization or emulsion techniques cannot be used in applications where the size distribution of the particles must be very narrow. For example, when using particles in blood purification applications, small particles must be avoided as small particles could be caught by the carrier fluid and would result in contamination of the purified material. Of course a narrow particle size distribution improves performances of particles in many applications, including chromatographic applications.
Operating variables that influence droplet size produced from atomizer wheels and hence particle size include speed of rotation, wheel diameter, wheel design, feed rate, viscosity of feed and air, density of feed and air and surface tension of feed.
The atmosphere within which a particle passes is important in order to avoid reduction of pore size. In particular, humidity and temperature control avoids particle desiccation during polymerization and gelling stages. Particle desiccation reduces pore size. It is desirable to have a machine and process to produce particles using centrifugal action in such a manner as that the particles have a narrow particle size distribution with both high porosity and flow.
Lengthy consideration of prior art devices and processes has identified a number of factors that may be responsible for the wider size distribution of particles. Such factors include interruptions on the wheel surface that may impede radial acceleration of the particle solution and adhesion to the surface of the wheel; lack of adequate temperature control on the atomizer wheel that may result in changes in feed viscosity and particle structure; and uncontrolled airflow patterns at the perimeter of the atomizer wheel that may result in particle twinning due to collisions between particles prior to gelation and in undesired drying of the particles due to a modification in their path down from the wheel to the collecting liquid.